竹节香附素A通过ULK1/Atg13及ROS途径调控肝癌细胞自噬的作用研究  

Study on the effect of raddeanin A in regulating autophagy of hepatocellular carcinoma cells through ULK1/Atg13 and ROS pathway

作　　者：赵利[1] 马向明[1] 王瑛[1] 吉瑞更 付庆江[1] 曹立瀛[1] Zhao Li;Ma Xiangming;Wang Ying;Ji Ruigeng;Fu Qingjiang;Cao Liying(Department of Hepatobi liary Surgery,Kailuan General Hospital,Hebei Province,Tangshan 063001,China)

机构地区：[1]开滦总医院肝胆外科,唐山063001

出　　处：《疑难病杂志》2020年第10期999-1004,共6页Chinese Journal of Difficult and Complicated Cases

基　　金：河北省医学科学研究重点课题计划项目(20181712);唐山市科学技术研究与发展计划项目(18130237a)。

摘　　要：目的探讨竹节香附素A通过自噬激活激酶1(ULK1)/自噬相关蛋白13(Atg13)及活性氧(ROS)途径调控肝癌细胞自噬的作用。方法2019年8—9月于开滦总医院实验室进行实验,设肝癌HepG2细胞组、5-氟尿嘧啶组(80 mg/μl)、竹节香附素A低剂量组(100 mg/μl)、竹节香附素A高剂量组(200 mg/μl),4组每孔设6个平行样,培养72 h,培养结束后,测定细胞存活率、自噬、凋亡率,检测ULK1、Atg13 mRNA和蛋白水平,2,7-二氯二氢荧光素二乙酸酯(DCFH-DA)测定ROS水平。结果与肝癌HepG2细胞组比较,5-氟尿嘧啶组及竹节香附素A低、高剂量组HepG2细胞OD值、存活率降低(P<0.01),自噬小体、HepG2细胞凋亡率、ULK1、Atg13 mRNA和蛋白表达、ROS水平升高(P<0.01);与5-氟尿嘧啶组比较,竹节香附素A低、高剂量组细胞OD值、存活率升高(t/P=21.214/0.000、19.547/0.000、18.547/0.000、25.478/0.000),自噬小体、凋亡率、ULK1、Atg13 mRNA和蛋白表达水平、ROS水平降低(t/P=16.325/0.000、22.547/0.000、9.874/0.000、16.589/0.000、13.654/0.000、14.654/0.000、16.589/0.000、21.247/0.000、21.687/0.000、18.324/0.000、18.541/0.000、19.587/0.000、19.654/0.000、24.052/0.000);与竹节香附素A低剂量组比较,竹节香附素A高剂量组细胞OD值、存活率降低(t/P=16.547/0.000、23.547/0.000),自噬小体、凋亡率、ULK1、Atg13 mRNA和蛋白表达水平、ROS水平升高(t/P=15.696/0.000、10.654/0.000、14.697/0.000、18.324/0.000、17.024/0.000、19.634/0.000、18.547/0.000)。结论竹节香附素A能明显抑制肝癌细胞增殖,促进肝癌细胞自噬及凋亡;其机制与竹节香附素A激活ULK1/Atg13及ROS通路有关。Objective To explore the role of Raddeanin A in regulating the autophagy of liver cancer cells through autophagy activated kinase 1(ULK1)/autophagy-related protein 13(Atg13)and reactive oxygen species(ROS)pathways.Methods The experiment was carried out in the laboratory of Kailuan General Hospital from August to September 2019,including the liver cancer HepG2 cell group,5-fluorouracil group(80 mg/μl),Raddeanin A low-dose group(100 mg/μl),Raddeanin A high-dose group(200 mg/μl),6 parallel samples in each well of 4 groups were cultured for 72 hours.After the culture,the cell survival rate,autophagy and apoptosis rate were measured,and ULK1,Atg13 mRNA and Protein level,2,7-dichlorodihydrofluorescein diacetate(DCFH-DA)measures ROS level.Results Compared with the HepG2 cell group of liver cancer,the 5-fluorouracil group and the low-dose and high-dose group of Raddeanin A of HepG2 cell OD value and survival rate decreased(P<0.01),autophagosome,HepG2 cell apoptosis rate,ULK1,Atg13 mRNA and protein expression,and ROS levels increased(P<0.01);compared with the 5-fluorouracil group,the cell OD value and survival rate of the low and high doses of Raddeanin A was increased(t/P=21.214/0.000,19.547/0.000,18.547/0.000,25.478/0.000),autophagosomes,apoptosis rate,ULK1,Atg13 mRNA and protein expression levels,and ROS levels decreased(t/P=16.325/0.000,22.547/0.000,9.874/0.000,16.589/0.000,13.654/0.000,14.654/0.000,16.589/0.000,21.247/0.000,21.687/0.000,18.324/0.000,18.541/0.000,19.587/0.000,19.654/0.000,24.052/0.000);compared with Raddeanin A low-dose group,the cell OD value and survival rate of the high-dose group decreased(t/P=16.547/0.000,23.547/0.000),autophagosomes,apoptosis rate,ULK1,Atg13 mRNA and protein expression levels,and ROS levels increased(t/P=15.696/0.000,10.654/0.000,14.697/0.000,18.324/0.000,17.024/0.000,19.634/0.000,18.547/0.000).Conclusion Raddeanin A can significantly inhibit the proliferation of liver cancer cells and promote autophagy and apoptosis of liver cancer cells;its mechanism is related to the activ

关 键 词：竹节香附素A ULK1/Atg13通路 活性氧 肝癌细胞 自噬 

分 类 号：R285[医药卫生—中药学]